In the course of studying inflammatory muscle diseases, we have encountered patients with other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and Type II Glycogen Storage Disease (acid alpha-glucosidase deficiency - also known as acid maltase deficiency or Pompe Disease). Our experience of treating Pompe mice (a knockout strain) and the experience in trials in humans, carried out in part under a CRADA with Genzyme, the company producing the recombinant drug, demonstrated that the drug is highly effective in clearing glycogen stored in cardiac muscle cells and only poorly effective for skeletal muscle cells. This failure of skeletal muscle response to recombinant enzyme in Pompe Syndrome has become a major focus of our attention and has led us into active new areas of cell biology that in themselves add great interest to our activities. The recombinant human enzyme itself is structurally and enzymatically adequate as established by glycogen clearance in the heart and partial clearance in some skeletal muscle cells. In Pompe mice, only Type I, slow-twitch, oxidative skeletal muscle cells are cleared. We had earlier explored differences between type I and type II (fast-twitch, glycolytic) cells in both normal and Pompe mice that might point to an explanation. Although Pompe mice had more early endosomal Rab5 protein and late endosomal/lysosomal LAMP-2 protein than WT mice in all tissues, Type I and II fibers did not differ. But three proteins involved in endocytosis - the cation-independent mannose-6-phosphate receptor, clathrin, and the adapter protein complex AP-2 were markedly lower in Type II muscles, the ratio of AP-2 isotypes differed. There are diminished levels of other trafficking proteins in Type II fibers: TfR, GGA2, and AP-1. Most strikingly, large autophagic areas were present uniformly in type II fibers in Pompe mice but present at the same low level in Pompe Type I fibers as in WT mice. We moved to studies of myoblasts and to studies of single fibers taken from Type I predominant (soleus) or Type II predominant (gastrocnemius) muscles. This allowed us to observe the various vacuolar compartments within single cells by confocal microscopy, using suitably labeled markers either with fixed or living cells. Vesicular movement in cultured myoblasts was markedly reduced in Pompe compared to WT myoblasts and there was a reduced number of lysosomes or late endosomes with a normal pH and the presence of an alkaline endosomal population. In addition, we noted remarkable differences between Type I & II Pompe in the distribution of lysosomes: in Type I fibers, lysosomes are arranged in long stretches leading to a tube-like structure, and Golgi markers neighbor lysosomes in Pompe as in WT fibers. By contrast, in Type II Pompe fibers, lysosomes are distributed throughout the fiber, and the Golgi markers are less regular and less likely to neighbor lysosomes. Most strikingly, the large areas of autophagic build-up, which may occupy close to half the diameter of a fiber, are usually centrally located, and disrupt the microtubular network and the contractile apparatus itself. With the Light Imaging Section and using second harmonic generation combined with two-photon excited fluorescence (E. Ralston and K. Zaal) we have noted early accumulation of lipofuscin in autophagic areas, presumably reflecting oxidative stress. In the past year, we have successfully developed long-term lines of Pompe myoblasts transfected with CDK4 that retain the ability to mature and fuse into myotubes. Using these myotubes treated with RNAi for the autophagic protein atg 7 and using primary myotubes cultured from Pompe mice with knockout in skeletal muscle of the autophagic protein atg 5, we have noted that glycogen accumulation in lysosomes is unimpaired, clearly showing that macroautophagy is not the way that glycogen is delivered to lysosomes, overturning a long held belief. In collaboration Dr. Raben and Dr. Takikita, E Richard and G Drouillard from France explored the consequences of substrate deprivation by using shRNA for glycogenin or for glycogen synthase in culture or delivering shRNA for glycogen synthase intramuscularly in mice using an AAV vector to Pompe mice. Both methods of blocking glycogen synthesis reduced glycogen accumulation in muscle cells (in press, 2008). To explore the role of disordered autophagy in causing muscle damage, we developed double KO mice lacking both the essential autophagy gene ATG5 and GAA. We confirmed that macroautophagy is not required to bring glycogen to lysosomes. Further, we found that in Pompe Disease there is induction of autophagy but a local functional deficiency because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may be responsible for the profound damage seen (in press, 2008). We have recently begun attempts to convert mouse fast twitch to slow twitch fibers by crossing Pompe mice to mice that are transgenic for PGC1alpha. We postulate that glycogen would then be able to be cleared from the fibers. In parallel we are trying to achieve the same end chemically by using SIRT1 agonists such as resveratrol and related compounds. Experiments with Drs. Leepo Yu and Robert Horowits to measure contractility of Pompe muscle fibers directly are in process.